The specificity and potency of targeted gene knockdown through RNA interference (RNAi) has generated tremendous excitement for utilizing this ubiquitous pathway in the clinic. Our studies investigate RNAi as a potential microbicide to target sexually transmitted diseases including HSV-2 and HIV-1. HSV-2 infects ~ 20% of adults in the US and up to 90% in parts of sub-Saharan Africa. It is associated with high morbidity and there is no cure or prophylactic treatment. HSV-2 is also a major co-factor for HIV-1. Therefore, a microbicide to HSV-2 could also have a significant impact on slowing HIV-1 transmission. Using a mouse model of vaginal HSV-2 infection, we were the first group to show that vaginal application of siRNAs targeting HSV-2 viral genes or host-encoded viral entry receptor genes could protect mice from HSV-2 infection. These findings were encouraging, but we encountered issues that would need to be addressed for siRNAs to be developed for clinical use. Problems included toxicity of a cationic lipid used to complex the siRNAs, transient protection conferred by virus-specific siRNAs, and siRNA instability in vaginal washes. Each of these problems was addressed in a follow-up study. In this proposal we will undertake a comprehensive analysis of HSV-2 gene targeted siRNAs to maximize their specific silencing ability, and minimize any off-target effects. Using the murine HSV-2 infection model we will determine whether: (i) immune responses, induced either by the siRNA or through viral challenge affect siRNA-mediated protection; (ii) enhanced incorporation of siRNA into RISC can result in more potent or durable protection; and (iii) efficient siRNA uptake and intracellular localization in the vaginal mucosa requires the expression of a specific receptor.